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Background: Several different molecular methods have been developed that are capable of detecting HIV-1 in clinical specimens with different levels of sensitivity and specificity. This article describes the results of a reliability study on the development and application of a new real-time TMA method for isothermal detection of HIV-1. Materials and Methods: In this ex Primental study, the molecular beacon primer and probe set were designed for a 176-base-pair region of HIV-1 pol gene using a specialized software. Logarithmic serial dilutions from 10-107 copies of an in-vitro transcribed RNA were used for determination of the analytical sensitivity of the assay. Clinical specimens that had previously been evaluated positive or negative by a valid commercial assay were used for assessing the clinical sensitivity and specificity of the assay. Results: The analytical and clinical sensitivities of the assay were determined 500 copies/ml and 93.3%, respectively. The primers and the probe were HIV-1 specific and no cross-reaction was observed with other blood-borne viruses and human genome bioinformatically. The clinical specificity of the developed real-time TMA assay was examined experimentally using 20 negative samples and determined to be 100%. Conclusion: The developed real-time TMA assay can be used as an appropriate tool for the rapid and isothermal detection of HIV-1 in patients' blood and plasma samples.